The impact of antibiotic induction on virulence and antibiotic resistance in Klebsiella pneumoniae

Background: Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections, classified into carbapenem-sensitive and carbapenem-resistant strains. Understanding the virulence factors and antibiotic resistance of these strains is essential for effective clinical management.

Objective: This study compared the virulence genes and antibiotic resistance profiles of 50 carbapenem-sensitive Klebsiella pneumoniae (CSKP) strains and 50 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains, examining their expression under antibiotic pressure and the mechanisms contributing to their pathogenicity.

Methods: Virulence genes (rmpA, rmpA2, iucA, iutA, Peg-344, ybts, iroB) were detected in both strains using polymerase chain reaction (PCR). Antibiotic susceptibility testing established minimum inhibitory concentrations (MICs) for key antibiotics. Gene expression analysis was performed with quantitative reverse transcription PCR (qRT-PCR) after 10 days of antibiotic exposure.

Results: CSKP strains exhibited significantly higher positivity rates for virulence genes compared to CRKP strains. CRKP strains predominantly expressed resistance genes KPC, SHV, and CTX-M3, whereas no resistance genes were found in CSKP. Antibiotic susceptibility tests showed increased MICs, particularly for ciprofloxacin and imipenem, following antibiotic induction. CSKP demonstrated elevated expression of rmpA and rmpA2, while CRKP showed increased expression of SHV, and KPC after antibiotic exposure.

Conclusion: This study highlights the intricate relationship between virulence and resistance in Klebsiella pneumoniae. CSKP strains show strong virulence factor expression, while CRKP strains adapt to antibiotic pressure through altered gene expression patterns. These findings underscore the urgent need for continuous surveillance and innovative therapeutic strategies to combat multidrug-resistant Klebsiella pneumoniae infections

Methods
We performed conventional PCR for virulence and resistance genes in bacterial strains. DNA was extracted by cell lysis in buffer and heating (100 °C, 10 min). After centrifugation (12,000 rpm, 5 min), the supernatant with ~100 ng/µL DNA was collected.
PCR (25 µL) had 2.0 µL DNA, 1.0 µL each of primers, 12.5 µL 2X Taq MasterMix, and 8.5 µL water. Thermal cycling: 94°C 3 min, 30 cycles (94°C 30 s, 55°C 30 s, 72°C 1 min), and 72°C 10 min. Agarose gel electrophoresis analyzed amplified products. Eight carbapenem - sensitive and - resistant strains were selected.
Antibiotic Induction Experiment
Eight carbapenem-resistant Klebsiella pneumoniae (CRKP) strains (with resistance and virulence genes) and eight carbapenem-sensitive Klebsiella pneumoniae (CSKP) strains (same virulence genes) strains were chosen. Strains were inoculated into LB broth and incubated at 37°C to logarithmic phase. The antibiotic - induction protocol was adjusted (Wistrand - Yuen et al., 2018). Induction: Day 1, 1/2 MIC; Day 2, 1 MIC; Day 3, 2 MIC, increasing for 10 days to 512 MIC or 64 μg/mL (if initial MIC > 64 μg/mL). Daily treatment with antibiotics to assess susceptibility and resistance changes.
 Real - Time Quantitative PCR (qPCR) Analysis
Three time points (baseline, Day 4, Day 7, Day 10) were selected after antibiotic - induction (Gaudin et al., 2013; Viers et al., 2014). Total RNA was isolated using a kit (Bioteke, Shanghai).
After RNA extraction, reverse transcription generated cDNA. qPCR with SYBR Green Master Mix and primers measured gene expression before and after antibiotic exposure. qPCR protocol: 95°C 3 min, 40 cycles (95°C 3 s, 60°C 30 s).
Primer sequences are in Supplementary Table 2. Each gene was analyzed in triplicate. The 16S rRNA gene was the internal control, and data were quantified by 2 - ΔΔCT method.